Engineering of a Lectibody Targeting High-Mannose-Type Glycans of the HIV Envelope.

High-mannose-type glycans (HMGs) are aberrantly enriched on HIV envelope glycoproteins. However, there is currently no drug selectively targeting HIV-associated HMGs. Here, we describe a novel HMG-targeting "lectibody," a recombinant Fc-fusion protein comprising human IgG1 Fc and a novel actinohivin lectin variant (Avaren) obtained by structure-guided modifications for improved overall surface charge properties (AvFc). AvFc was engineered and produced using a rapid and scalable plant-based transient overexpression system. The lectibody exhibited potent antiviral activity against HIV-1 groups M and O primary viruses, as well as HIV-2 and simian immunodeficiency virus (SIV) strains, without affecting normal human blood cells. Furthermore, the lectibody induced Fc-mediated cell killing activity against HIV-1-infected cells and selectively recognized SIVmac239-infected macaque mesenteric lymph node cells in vitro. AvFc showed an extended serum half-life in rats and rhesus macaques, while no discernible toxicity was observed upon repeated systemic dosing in mice. These results highlight AvFc's potential as a biotherapeutic targeting HIV-associated HMGs of cell-free virions, as well as productively infected cells, providing a foundation for new anti-HIV strategies. Efficient and cost-effective bioproduction in greenhouse facilities may open unique possibilities for further development of AvFc.

A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D.

We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 µM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.

Reducing infectivity of HIV upon exposure to surfaces coated with N,N-dodecyl, methyl-polyethylenimine.

The infectivity of high-titer, cell-free HIV in culture media and human milk is rapidly reduced upon exposure to polyethylene slides painted with the linear hydrophobic polycation N,N-dodecyl,methyl-polyethylenimine (DMPEI). Accompanying viral p24 protein and free viral RNA analysis of solutions exposed to DMPEI-coated surfaces suggests that virion attachment to the polycationic surface and its subsequent inactivation are the likely mechanism of this phenomenon.

HIV-1 neutralization profile and plant-based recombinant expression of actinohivin, an Env glycan-specific lectin devoid of T-cell mitogenic activity.

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1's AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide.

Peptide sugar mimetics prevent HIV type 1 replication in peripheral blood mononuclear cells in the presence of HIV-positive antiserum.

Cells of the immune system express a number of receptors that bind carbohydrate ligands. We questioned whether peptide mimetics of these ligands will activate phagocytic cells and thereby enhance an antiviral response. Short peptide sequences were identified by computational modeling of docking to glycan-specific lectins, selected as receptor analogs, and incorporated into quadravalent structures by peptide synthesis. A peptide with the sequence HPSLK bound to several lectins specific for monosaccharides and to lectins specific for Neu5Ac-Gal-containing complex glycans, whereas a longer sequence, NPSHPLSG, bound only lectins specific for the more complex glycans. In cultures of peripheral blood mononuclear cells (PBMCs) these peptides stimulated phagocytosis of opsonized microspheres. The peptides inhibited replication of HIV-1 in PBMC cultures by 20-80% at concentrations between 1 nM and 1 muM but inhibited replication 100% in the presence of diluted HIV-positive antiserum that alone inhibited replication by 30%. HPSLK caused about 50% loss of viability of cells at 1 mM, a concentration 10(6)-fold higher than an effective inhibitory concentration, but no toxicity was observed with NPSHPLSG. These results demonstrated that peptidomimetics of glycan ligands of cellular receptors are effective in activating phagocytosis, which may be a factor in providing complete inhibition of HIV-1 replication in vitro.

Serologic diagnosis of West Nile and St. Louis encephalitis virus infections in domestic chickens.

Adult domestic chickens were infected with West Nile virus (WNV) or St. Louis encephalitis virus (SLEV) and challenged with homologous or heterologous virus at 21 or 56 days postinfection (dpi). Sera were collected at selected time points after infection and assayed by enzyme immunoassay (EIA), plaque reduction neutralization test (PRNT), and a Western blot (WB) alternative to PRNT. EIA results were sensitive and accurate (few false positives) but not specific, requiring a confirmatory test to determine virus infection history. PRNT results generally were specific until challenge, after which test results were frequently equivocal and inadequate to determine first or second infecting virus. WB results confirmed the serologic cross-reactivity between WNV and SLEV envelope protein. Non-structural protein 1 and pre-membrane protein reactivities were highly specific for WNV during SLEV infection, but less specific for SLEV during WNV infection. WB and PRNT specificities were similar for both viruses from 6 to 14 dpi, and sensitivities to WNV were virtually identical.

Towards covalent vaccination: improved polyclonal HIV neutralizing antibody response induced by an electrophilic gp120 V3 peptide analog.

Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) approximately 50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pretreatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines.

Inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long-wavelength ultraviolet light.

BACKGROUND: Viral contamination of platelet (PLT) concentrates can result in transfusion-transmitted diseases. A photochemical treatment (PCT) process with amotosalen-HCl and long-wavelength ultraviolet light (UVA), which cross-links nucleic acids, was developed to inactivate viruses and other pathogens in PLT concentrates. STUDY DESIGN AND METHODS: High titers of pathogenic or blood-borne viruses, representing 10 different families, were added to single-donor PLT concentrates containing 3.0 x 10(11) to 6.0 x 10(11) PLTs in approximately 300 mL of 35 percent plasma and 65 percent PLT additive solution (InterSol). After PCT with 150 micromol per L amotosalen and 3 J per cm(2) UVA, residual viral infectivity was assayed by sensitive cell culture or animal systems. RESULTS: Enveloped viruses were uniformly sensitive to inactivation by PCT whereas nonenveloped viruses demonstrated variable inactivation. Log reduction of enveloped viruses for cell-free HIV-1 was >6.2; for cell-associated HIV-1, >6.1; for clinical isolate HIV-1, >3.4; for clinical isolate HIV-2, >2.5; for HBV, >5.5; for HCV, >4.5; for DHBV, >6.2; for BVDV, >6.0; for HTLV-I, 4.2; for HTLV-II, 4.6; for CMV, >5.9; for WNV, >5.5; for SARS-HCoV, >5.8; and for vaccinia virus, >4.7. Log reduction of nonenveloped viruses for human adenovirus 5 was >5.2; for parvovirus B19, 3.5->5.0; for bluetongue virus, 5.6-5.9; for feline conjunctivitis virus, 1.7-2.4; and for simian adenovirus 15, 0.7-2.3. CONCLUSION: PCT inactivates a broad spectrum of pathogenic, blood-borne viruses. Inactivation of viruses in PLT concentrates with amotosalen and UVA offers the potential to prospectively prevent the majority of PLT transfusion-associated viral diseases.

Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques.

The identification of HIV envelope structures that generate broadly cross-reactive neutralizing antibodies is a major goal for HIV-vaccine development. In this study, we evaluated one such structure, expressed as either a gp120-CD4 or a gp140-CD4 complex, for its ability to elicit a neutralizing antibody response. In rhesus macaques, covalently crosslinked complexes of soluble human CD4 (shCD4) and HIV-1(IIIB) envelope glycoproteins (gp120 or gp140) generated antibodies that neutralized a wide range of primary HIV-1 isolates regardless of the coreceptor usage or genetic subtype. Ig with cross-reactive neutralizing activity was recovered by affinity chromatography with a chimeric single-chain polypeptide containing sequences for HIV(BaL) gp120 and a mimetic peptide that induces a CD4-triggered envelope structure. These results suggest that covalently crosslinked complexes of the HIV-1 surface envelope glycoprotein and CD4 elicit broadly neutralizing humoral responses that, in part, may be directed against a novel epitope(s) found on the HIV-1 envelope.